Molecular mechanisms of building blocks of life towards medicinal applications

Abstract

Membrane and membrane-spanning proteins, and non-coding RNA are biomolecules to play central roles in beginning of life and distinguishing higher-order eukaryotes. We have determined the structures of membrane protein-lipids complexes and non-coding RNA-protein complexes by X-ray crystallography and Cryo-EM single particle analysis, and combined with complementary functional analyses, elucidate their molecular mechanisms at atomic resolutions, to promote creating drugs and medical technologies with two venture companies.

Membrane and membrane-spanning proteins, and non-coding RNA are biomolecules to play central roles in beginning of life and distinguishing higher-order eukaryotes. We have determined the structures of membrane protein-lipids complexes and non-coding RNA-protein complexes by X-ray crystallography and Cryo-EM single particle analysis, and combined with complementary functional analyses, elucidate their molecular mechanisms at atomic resolutions, to promote creating drugs and medical technologies with two venture companies.

Membrane channels and GPCRs have central roles in neural transmission in brain, which defines higher-order eukaryotes. Especially, we promote structural biology to elucidate how physical sensors open the channel upon perception of light, sound, temperature and voltage. Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity by light illumination. We solved the first crystal structure of channelrhodopsin at 2.3 Å resolution, showing a cation conducting pore with two gates possibly regulated by the conformational transition of the light-harvesting retinal (Nature, 2012). Based on the structure, we created blue-shifted ChR variant useful for neuroscience (Nat. Commun., 2015). We also solved the crystal structure of the most red-shifted ChR, Chrimson, to elucidate the red-shift mechanism and succeeded in the creation of an even more red-shifted mutant with higher kinetics (Nat. Commun., 2019). However, the detailed molecular events underlying channel gating remain yet unknown. Recently, we succeeded in the time-resolved crystallography of ChR using X-ray free electron laser (XFEL), showing that the twisting of retinal triggers the movement of the transmembrane helices (outward shift of TM3 and local deformation in TMs6 and 7) to open the channel (eLife, 2021).

Furthermore, by using Cryo-EM single particle analysis, we have elucidated molecular mechanisms at atomic resolutions of sound-amplifying motor prestin, heat-sensor TRP channel (Nat. Struct Mol. Biol., 2020), multi-subunit voltage gated potassium channel, Kv4.2 supramolecular complex (Nature, 2021) and so on. We also solved the Cryo-EM structures of P4 flippase in 6 intermediate states to elucidate how it flips phosphatidylserine to protect normal cells from apoptotic phagocytosis (Science, 2019). Furthermore, we solved 5 GPCRs in complex with their ligands and coupling trimeric GTPase to elucidate the molecular mechanisms of ligand capturing and recognition, and G-protein activation/inactivation (Nat. Struct Mol. Biol., 2020, 2021; Mol. Cell, 2021, 2021, 2021). As dysfunction of these membrane proteins cause severe diseases, we established new venture company, Curreio to create new drugs based on the Cryo-EM structures and their molecular mechanisms.

Genome editing has been expected as the state-of-the-art technology for gene therapy, but CSRIPR-Cas nuclease still has several problems to hamper their clinical application. First, molecular weights of Cas nucleases are too large for their genes to be packaged in virus vectors to introduce into eukaryotic cells. Second, CRISPR-Cas nucleases strictly recognize PAM sequence just downstream of the target sequence in genome, which prevents CRISPR-Cas from freely targeting arbitrary region in the genome. Third, CRISPR-Cas nucleases mistakenly edit incorrect genome region, known as off-target activity. To overcome these problems, we solved Cas9 proteins from 5 species and Cas12 proteins from 5 species, in complexes with guide RNA and target DNA (Cell, 2014; Cell, 2015; Cell, 2016; Cell, 2016; Mol. Cell, 2017; Mol. Cell, 2017; Nat. Commun., 2019; Mol. Cell, 2021). We also solved the binary and ternary complex structures of miniature Cas13, which paves the way for developing mRNA editing technology. Based on these structures, we succeeded in engineering Cas9 variants that recognize a simple PAM (a single guanine) (Science, 2018). Based on our achievement of “super Cas effectors with miniature size, targeting freedom and high specificity, we established a venture company, Modalis (IPO, 2020) to complete gene therapy of genetic rare diseases.